Sara Fossati

Therapy‐induced antitumor vaccination by targeting tumor necrosis factor‐α to tumor vessels in combination with melphalan

Treatment of tumor‐bearing mice with mouse (m)TNF‐α, targeted to tumor vasculature by the anti‐ED‐B fibronectin domain antibody L19(scFv) and combined with melphalan, induces a therapeutic immune response. Upon treatment, a highly efficient priming of CD4+ T cells and consequent activation and maturation of CD8+ CTL effectors is generated, as demonstrated by in vivo depletion and adoptive cell transfer experiments. Immunohistochemical analysis of the tumor tissue demonstrated massive infiltration of CD4+ and CD8+ T cells 6 days after treatment and much earlier in the anamnestic response to tumor challenge in cured mice. In fact, the curative treatment with L19mTNF‐α and melphalan resulted in long‐lasting antitumor immune memory, accompanied by a mixed Th1/Th2‐type response and significant in vitro tumor‐specific cytolytic activity. Finally, the combined treatment reduced the percentage and absolute number of CD4+CD25+ regulatory T cells in the tumor‐draining lymph nodes of mice responding to therapy, and this was associated with the establishment of protective immunity. These findings pave the way for alternative therapeutic strategies based on the targeted delivery of biological and pharmacological cytotoxic compounds that not only kill most of the tumor cells but, more importantly, trigger an effective and long‐lasting antitumor adaptive immune response.

A novel human fibronectin cryptic sequence unmasked by the insertion of the angiogenesis‐associated extra type III domain B

The angiogenesis‐associated extra‐domain B (EDB) of fibronectin (FN) is a complete type III repeat of 91 amino acids. Its expression is modulated by the alternative splicing pattern of the FN pre‐mRNA. FN containing the EDB (B‐FN) is undetectable in tissues of healthy adults, with rare exceptions such as the female reproductive system where tissue remodeling and angiogenesis are recurrent physiological processes. On the contrary, B‐FN is expressed at high levels in neoplastic tissues and during angiogenesis; consequently, it is considered an excellent marker of angiogenesis. Here, we report on a novel FN cryptic sequence, localized on the FN type III repeat 8 (immediately downstream of the EDB) that is unmasked by the insertion of the EDB. This sequence is specifically recognized by the high‐affinity monoclonal antibody, C6, that selectively recognizes B‐FN by means of ELISA, immunohistochemical and Western blot assays. The variable regions of C6 were cloned and a divalent covalently linked mini‐antibody was generated. Biodistribution studies using the radioiodinated C6 mini‐antibody on tumor‐bearing mice demonstrated an efficient tumor targeting. This antibody represents a new tool for the study of the potential biological functions of hindered sequences that the inclusion of the EDB renders accessible, and likewise makes its epitope an additional angiogenesis target.

Identification and Expression of Acetylcholinesterase in Octopus vulgaris Arm Development and Regeneration: a Conserved Role for ACHE?

Acetylcholinesterase (ACHE) is a glycoprotein with a key role in terminating synaptic transmission in cholinergic neurons of both vertebrates and invertebrates. ACHE is also involved in the regulation of cell growth and morphogenesis during embryogenesis and regeneration acting through its non-cholinergic sites. The mollusk Octopus vulgaris provides a powerful model for investigating the mechanisms underlying tissue morphogenesis due to its high regenerative power. Here, we performed a comparative investigation of arm morphogenesis during adult arm regeneration and embryonic arm development which may provide insights on the conserved ACHE pathways. In this study, we cloned and characterized O. vulgaris ACHE, finding a single highly conserved ACHE hydrophobic variant, characterized by prototypical catalytic sites and a putative consensus region for a glycosylphosphatidylinositol (GPI)-anchor attachment at the COOH-terminus. We then show that its expression level is correlated to the stage of morphogenesis in both adult and embryonic arm. In particular, ACHE is localized in typical neuronal sites when adult-like arm morphology is established and in differentiating cell locations during the early stages of arm morphogenesis. This possibility is also supported by the presence in the ACHE sequence and model structure of both cholinergic and non-cholinergic sites. This study provides insights into ACHE conserved roles during processes of arm morphogenesis. In addition, our modeling study offers a solid basis for predicting the interaction of the ACHE domains with pharmacological blockers for in vivo investigations. We therefore suggest ACHE as a target for the regulation of tissue morphogenesis.

Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins

We report a novel strategy to engineer and express stable and soluble human recombinant polyvalent/polyspecific fusion proteins. The procedure is based on the use of a central skeleton of uteroglobin, a small and very soluble covalently linked homodimeric protein that is very resistant to proteolytic enzymes and to pH variations. Using a human recombinant antibody (scFv) specific for the angiogenesis marker domain B of fibronectin, interleukin 2, and an scFv able to neutralize tumor necrosis factor-α, we expressed various biologically active uteroglobin fusion proteins. The results demonstrate the possibility to generate monospecific divalent and tetravalent antibodies, immunocytokines, and dual specificity tetravalent antibodies. Furthermore, compared with similar fusion proteins in which uteroglobin was not used, the use of uteroglobin improved properties of solubility and stability. Indeed, in the reported cases it was possible to vacuum dry and reconstitute the proteins without any aggregation or loss in protein and biological activity.

The making of an octopus arm

BackgroundMost of our current findings on appendage formation and patterning stem from studies on chordate and ecdysozoan model organisms. However, in order to fully understand the evolution of animal appendages, it is essential to include information on appendage development from lophotrochozoan representatives. Here, we examined the basic dynamics of the Octopus vulgaris arm’s formation and differentiation - as a highly evolved member of the lophotrochozoan super phylum - with a special focus on the formation of the arm’s musculature.ResultsThe octopus arm forms during distinct phases, including an early outgrowth from an epithelial thickening, an elongation, and a late differentiation into mature tissue types. During early arm outgrowth, uniform proliferation leads to the formation of a rounded bulge, which subsequently elongates along its proximal-distal axis by means of actin-mediated epithelial cell changes. Further differentiation of all tissue layers is initiated but end-differentiation is postponed to post-hatching stages. Interestingly, muscle differentiation shows temporal differences in the formation of distinct muscle layers. Particularly, first myocytes appear in the area of the future transverse prior to the longitudinal muscle layer, even though the latter represents the more dominant muscle type at hatching stage. Sucker rudiments appear as small epithelial outgrowths with a mesodermal and ectodermal component on the oral part of the arm. During late differentiation stages, cell proliferation becomes localized to a distal arm region termed the growth zone of the arm.ConclusionsO. vulgaris arm formation shows both, similarities to known model species as well as species-specific patterns of arm formation. Similarities include early uniform cell proliferation and actin-mediated cell dynamics, which lead to an elongation along the proximal-distal axis. Furthermore, the switch to an adult-like progressive distal growth mode during late differentiation stages is reminiscent of the vertebrate progress zone. However, tissue differentiation shows a species-specific delay, which is correlated to a paralarval pelagic phase after hatching and concomitant emerging behavioral modifications. By understanding the general dynamics of octopus arm formation, we established a basis for further studies on appendage patterning, growth, and differentiation in a representative of the lophotrochozoan super phylum.

Molecular Determinants of Cephalopod Muscles and Their Implication in Muscle Regeneration

The ability to regenerate whole-body structures has been studied for many decades and is of particular interest for stem cell research due to its therapeutic potential. Several vertebrate and invertebrate species have been used as model systems to study pathways involved in regeneration in the past. Among invertebrates, cephalopods are considered as highly evolved organisms, which exhibit elaborate behavioral characteristics when compared to other mollusks including active predation, extraordinary manipulation, and learning abilities. These are enabled by a complex nervous system and a number of adaptations of their body plan, which were acquired over evolutionary time. Some of these novel features show similarities to structures present in vertebrates and seem to have evolved through a convergent evolutionary process. Octopus vulgaris (the common octopus) is a representative of modern cephalopods and is characterized by a sophisticated motor and sensory system as well as highly developed cognitive capabilities. Due to its phylogenetic position and its high regenerative power the octopus has become of increasing interest for studies on regenerative processes. In this paper we provide an overview over the current knowledge of cephalopod muscle types and structures and present a possible link between these characteristics and their high regenerative potential. This may help identify conserved molecular pathways underlying regeneration in invertebrate and vertebrate animal species as well as discover new leads for targeted tissue treatments in humans.